This invention relates to agonist monoclonal antibodies (mAb) that bind to the Tie2 receptor and to the use of such antibodies for therapeutic purposes.
Angiogenesis or neovascularisation is the process of development of new or replacement blood vessels. It is a necessary and normal process by which the vasculature is established in the embryo. Angiogenesis does not occur, in general, in most normal adult tissues, exceptions being sites of ovulation, menses and wound healing.
Increasing blood supply is beneficial in many conditions such as ischemic conditions, resulting from stroke, myocardial infarction and other infarctions, and states of poor blood flow, resulting from diseases such as diabetes or peripheral vascular diseases. Vascular endothelial growth factor and basic fibroblast growth factor are factors that have been shown to promote angiogenesis. Recent work with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor has demonstrated that promotion of angiogenesis by these factors can have benefit in ischemic diseases and vascular disease (Bauters, C. et al., Circulation, 91: 2802-2809, 1995; Bauters, C. et al., Journal of Vascular Surgery, 21: 314-24, 1995; Isner, J. M. et al., Human Gene Therapy, 7: 959-988, 1996; Takeshita, S., et al., Biochemical and. Biophysical Research Communications, 227: 628-635, 1996; Tsurumi, Y., et al., Circulation, 96: 382-388, 1997).
In recent years, it has become clear that while angiogenesis is a complex, multicellular phenomena, specific ligands and their receptors play a key role. In particular, a combination of studies suggest that the Tie2 receptor and its ligand are important in angiogenesis.
Tie2 receptor has been located in endothelial cells of all forming blood vessels and in the endocardium of mouse embryos (Korhonen et al., Blood 80:2548-2555, 1992). Selective patterns of expression in endothelial cells during embryonic development has also been demonstrated (Schlaeger et al., Development 121:1089-1098, 1995).
In adult tissues, Tie mRNA cannot be observed in skin, except at sites of active wound healing where the proliferating capillaries in the granulation tissue contain abundant Tie mRNA (Korhonen et al., Blood 80:2548-2555, 1992). Further, Tie receptor is expressed in the vascular endothelium of metastasizing melanomas (Kaipainen et al., Cancer Res. 54:6571-6577, 1994). While Tie receptor expression is down-regulated in the established vasculature, it is upregulated in angiogenesis that occurs in the ovary during ovulation, in wounds and in tumor (breast, melanoma and renal cell carcinoma) vasculature, consistent with prevailing views that angiogenesis in the adult borrows from embryonic angiogenic mechanisms.
Homozygous mice with a Tie2 knockout, or carrying a transgene encoding a xe2x80x9cdominant-negativexe2x80x9d Tie2 receptor, confirmed that the Tie2 receptor is critical for embryonic development (Dumont et al., Genes Dev. 8:1897-1909, 1994; Sato et al., Nature 376:70‥74, 1995). Embryonic death in these mice occurred due to vascular insufficiency and there were dramatically reduced numbers of endothelial cells. Vasculogenesisxe2x80x94that is the differentiation of endothelial cells and the in situ formation of vesselsxe2x80x94appeared relatively normal in mice lacking Tie2. The subsequent sprouting and remodeling resulting in formation of vessel branches (angiogenesis) was drastically reduced in the Tie2 mutant mice embryos. This lack of sprouting and angiogenesis resulted in substantial growth retardation, particularly of the brain, neural tube and heart, resulting in lack of viability. These results exemplify the critical importance of Tie2 in angiogenesis. This is significant, as angiogenesis is regulated by a number of growth factors. Interestingly, Flk1 (VEGF receptor) knockout mice exhibit embryo lethal defects in vasculogenesis, that occur earlier than those of Tie2 disruption. Disruption of the Tie1 receptor yields a much different, and later, defective phenotype; the mouse embryo dies late in development due to hemorrhage resulting from defective integrity of an otherwise well-formed vasculature. Taken together, these studies suggest that the VEGF/Flk1 and Tie systems operate in sequential fashion, with Tie2 having a critical role in angiogenesis.
Recently, two ligands for the Tie2 receptor have been reported. Angiopoietin-1 binds and induces the tyrosine phosphorylation of Tie2 and its expression in vivo is in close proximity with developing blood vessels (Davis et al., Cell 87:1161-1169,1996). Mice engineered to lack Angiopoietin-1 display angiogenic deficits reminiscent of those previously seen in mice lacking Tie2 receptors, demonstrating that Angiopoietin-1 is a primary physiologic ligand for Tie2 and that Tie2 has critical in vivo angiogenic actions (Suri et al., Cell 87:1171-1180, 1996). Angiopoietin-2 was identified by homology screening and shown to be a naturally occurring antagonist for Tie2 receptors. Transgenic overexpression of Angiopoietin-2 disrupts blood vessel formation in the mouse embryo (Maisonpierre et al., Science 277:55-60, 1997). Together, these results support a role for Tie2 receptors in angiogenesis.
The Tie1 and Tie2 receptors are single-transmembrane, tyrosine kinase receptors (Tie stands for Tyosine kinase receptors with immunoglobulin and EGF homology domains). They are the only receptor tyrosine kinases, other than those receptors for VEGF, that are largely restricted to endothelial cells in their expression. Both have been cloned and reported by several groups (Dumont et al., Oncogene 8:1293-1301, 1993; Partanen et al., Mol. Cell Biol. 12:1698-1707, 1992; Sato et al., Proc. Natl. Acad. Sci. USA 90:9355-9358, 1993).
The Tie receptors are proteins of approximately 125 kDa, with a single putative transmembrane region. The extracellular domain of these receptors is uniquely divided into three regions that have a pattern of cysteine expression found in EGF-like domains; two regions that have some weak homology to and structural characteristics of immunoglobulin-like domains; and three regions with homology to the fibronectin III repeat structure. The intracellular portion of Tie2 is most closely related (xcx9c40% identity) to the kinase domains of FGF-R1, PDGF-R and c-kit. The intracellular portions of Tie2 contain all of the features of tyrosine kinases, including a GXGXXG ATP binding site consensus sequence and typical tyrosine kinase motifs (i.e., HRDLAARN and DFGL).
Based upon the importance of Tie2 receptors in angiogenesis, Tie2 receptor agonist activity is predicted to stimulate angiogenesis, providing disease-specific therapeutic effects. Clearly, there is a need to develop high affinity, potent agonist antibodies to the Tie2 receptor which will have sufficient activity to work in vivo at therapeutically acceptable concentrations.
Tie2 receptors are expressed on hematopoietic progenitors, including CD34+ cells, megakaryocyte progenitors and megakaryocyte-derived cell lines (Kukk et al., Brit J Haematol. 98:195-203, 1997; Batard et al., Blood 87:2212-2220, 1996). The Tie2 receptor has homology to other hematopoietic growth factor receptors such as c-kit (Chabot et al, Nature 335:88, 1988; Yarden et al., EMBO J 6:3341, 1987) and flk-2 (Matthews et al, Cell 65:1143, 1991). The expression of Tie2 decreased on more mature hematopoietic cells, although it was maintained on a significant fraction of cells during megakaryocytic differentiation (Batard). These data, taken together, suggest that Tie2 is a receptor for a hematopoietic growth factor acting on early progenitors and during differentiation of the megakaryocytic lineage.
One aspect of the present invention is a Tie2 receptor agonist antibody having the identifying characteristics of monoclonal antibody 15B8 or 13H10.
Another aspect of the invention is an antibody comprising a heavy chain variable region polypeptide as set forth in SEQ ID NO: 2 and a light chain variable region polypeptide as set forth in SEQ ID NO: 4 and isolated polynucleotides encoding them.
Another aspect of the invention is a heavy chain CDR having an amino acid sequence as set forth in SEQ ID NO: 5, 6 or 7.
Another aspect of the invention is a light chain CDR having an amino acid sequence as set forth in SEQ ID NO: 8, 9 or 10.
Another aspect of the invention is an antibody comprising a heavy chain variable region polypeptide as set forth in SEQ ID NO: 21 and a light chain variable region polypeptide as set forth in SEQ ID NO: 14 and isolated polynucleotides encoding them.
Another aspect of the invention is a heavy chain CDR having an amino acid sequence as set forth in SEQ ID NO: 15, 16 or 17.
Another aspect of the invention is a light chain CDR having an amino acid sequence as set forth in SEQ ID NO: 18, 19 or 20.
Another aspect of the invention is a hybridoma having the identifying characteristics of cell line 15B8 or 13H10.
Another aspect of the invention is a method for enhancing angiogenesis in a animal comprising administering an effective dose of an Tie2 receptor agonist antibody having the identifying characteristics of monoclonal antibody 15B8 or 13H10.
Another aspect of the invention is a method for enhancing endothelial cell survival in a mammal comprising administering an effective dose of a Tie2 receptor agonist antibody.
Another aspect of the invention is a method of enhancing hematopoietic or megakaryocytic cell proliferation in a mammal comprising administering an effective dose of a Tie2 receptor agonist antibody.